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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
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Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
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Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

Ying Shang1, Wentao Xu2, Yong Wang3

  • 1Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, Yunnan 650500, China; Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.

Food Chemistry
|August 3, 2017
PubMed
Summary
This summary is machine-generated.

A new pyrosequencing method enables qualitative detection of multiple genetically modified (GM) crops using a single primer. This technique also allows for semi-quantitative estimation of GM crop proportions in mixtures.

Keywords:
GM cropMultiplex qualitativePyrosequencingUP-M-PCR

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Accurate detection of genetically modified (GM) events is crucial for regulatory compliance and food safety.
  • Existing multiplex detection methods can be complex and require multiple primers.
  • There is a need for efficient and sensitive methods for identifying multiple GM targets simultaneously.

Purpose of the Study:

  • To develop and validate a novel multiplex qualitative detection method for GM crops using pyrosequencing.
  • To assess the sensitivity and specificity of the developed method.
  • To explore the potential for semi-quantitative analysis of GM event proportions.

Main Methods:

  • A universal primer-multiplex-PCR approach was utilized.
  • A single sequencing primer was employed for multiplex target detection.
  • Pyrosequencing technology was used for sequence-based detection.
  • dNTP dispensing order was optimized based on product sequences for qualitative detection.
  • Signal value calculation was used for semi-quantitative estimation.

Main Results:

  • The method successfully detected multiple GM crop events qualitatively with high sensitivity (as low as 1% in a mixture).
  • Detection required a minimal number of dNTP dispensing rounds (12 rounds, often as few as 3).
  • Signal values allowed for the estimation of the proportion of each GM event in a mixture, enabling semi-quantitative analysis.

Conclusions:

  • The novel pyrosequencing-based method provides an efficient and sensitive approach for qualitative multiplex detection of GM events.
  • The method demonstrates potential for semi-quantitative analysis, allowing for the estimation of individual GM event proportions in mixtures.
  • This technique offers a valuable tool for GM crop analysis and regulatory monitoring.